This invention relates to the field of enzyme inhibitors, especially inhibitors of histone acetyltransferases.
The molecular identification of a number of histone acetyltransferases (HATs) has led to new insights into the mechanisms of activation of gene expression (Mizzen and Allis, 1998; Stuhl, 1998). The members of this growing family include the important transcription factors p300/CBP and PCAF (Yang et al., 1996; Ogryzko et al., 1996; Bannister and Kouzarides, 1996). PCAF and p300/CBP can catalyze the acetylation of histones (Yang et al., 1996; Ogryzko et al., 1996; Bannister and Kouzarides, 1996) and other substrates (Wu and Roeder, 1997) and these HATs have been suggested to play differential roles in coactivation of gene expression. The HAT domain of PCAF appears to be involved in MyoD-dependent coactivation and differentiation, whereas that of p300 seems to be less important (Puri et al., 1997). A role for the acetyltransferase activity of PCAF was also suggested for transcriptional activation by the liganded retinoic acid receptor (RAR). The PCAF acetyltransferase domain but not that of CBP can assist retinoic acid induced transcription in cultured cells that are depleted of endogenous PCAF and CBP by antibody microinjection (Korzus et al., 1998). On the other hand, the acetyltransferase domain of CBP and not that of PCAF can contribute to CREB-activated transcription in PCAF- and CBP-depleted cells (Korzus et al., 1998). These experiments suggest differential requirements for PCAF and p300/CBP in the coactivation of various sequence-specific DNA binding transcriptional activators. However, none of these studies has established directly that acetyltransferase action or histone acetylation per se is involved in these activation processes.
Because of the possibility that PCAF and p300 proteins physically interact (Ogryzko et al., 1996), their relative contributions toward acetylation of substrates and gene activation is not generally known. While mutations in the active site regions of these enzymes can help clarify these issues, the effects of such mutations on altering protein structure and stability can complicate interpretations. Small molecules have been useful in elucidation of the general role of histone acetylation in transcription by blocking histone deacetylase (Taunton et al., 1996). It would be advantageous to apply active-site directed, specific, and potent synthetic inhibitors of individual HAT enzymes to dissect their relative roles in protein acetylation and transcription. Furthermore, there is a basis for expecting that the blockade of p300 HAT activity would have therapeutic potential in the treatment of certain cancers (Giles et al., 1998).
Prior to the molecular characterization of specific HAT enzymes, several polyamine-CoA conjugates were found to block HAT activities present in cell extracts (Cullis et al., 1982; Erwin et al., 1984). However the actual enzyme or enzymes inhibited have not been characterized. We have shown that one of these synthetic inhibitors (Cullis et al., 1982) potently blocks non-chromatin template mediated transcription and therefore would not be useful in the determination of the role of HAT activity in gene activation unpublished data.
PCAF belongs to a superfamily of GNAT (GCN-5 related N-acetyltransferases) acetyltransferases whose three-dimensional structures have recently been reported (Coon et al., 1995; Neuwald and Landsman, 1997; Wolf et al., 1998; Dutnall et al., 1998; Wybenga-Groot et al., 1999; Hickman et al., 1999a; Hickman et al., 1999b; Lin et al., 1999). Family members most likely catalyze acetyl transfer in a ternary complex containing enzyme, histone, and acetyl-CoA (De Angelis et al., 1998; Tanner et al., 1999). Bisubstrate analog inhibitors have proved successful for the GNAT family member serotonin N-acetyltransferase (Khalil et al., 1998; Khalil et al., 1999). Here we report on bisubstrate analog inhibitors and their effects on histone acetylation and transcription.
The invention in a general aspect is a histone acetyltransferase inhibitor. Such inhibitors are useful both as analytical reagents for studying the role of histone acetyltransferases in the regulation of gene expression. They are also useful for inhibiting acetyltransferase in diseased cells that overexpress such acetyltransferase.
In a particular embodiment of the invention, the inhibitor is Coenzyme A (CoA) covalently linked (preferably via a xe2x80x94COxe2x80x94 bridge to a lysine xcex5-amino group) to lysine or a polypeptide comprising lysine. Inhibitors that are specific for p300 acetyltransferase are those in which the CoA is linked to lysine or a very short polypeptide (2 to 6 amino acids) comprising lysine. Such inhibitors will inhibit p300 acetyltransferases (xe2x80x9cp300 inhibitorsxe2x80x9d) more significantly (at least 100 times as much) than they inhibit PCAF acetyltransferases. Inhibitors that are specific for PCAF acetyltransferases (xe2x80x9cPCAF inhibitorsxe2x80x9d) are those in which the CoA is linked to lysine in longer polypeptides (8 or more amino acids.) Such inhibitors will inhibit PCAF acetyltransferases (xe2x80x9cPCAF inhibitorsxe2x80x9d) more significantly (at least 100 times as much) than they inhibit p300 acetyltransferases.
In another aspect, the invention is histone acetylase inhibitors that will inhibit transcription of a histone-associated DNA sequence more strongly than the identical DNA sequence not associated with histones (especially, naked DNA). Such inhibitors are the p300 inhibitors and PCAF inhibitors.
In another general aspect, the invention is the process of administering a histone acetyltransferase inhibitor to a host, the host being an animal or human. Such a process is done for therapeutic purposes in cases where it is beneficial to the host to have a histone acetyltransferase inhibited. That is the case, for example, in certain types of cancers. It can also be the case in certain gene therapy protocols.
A preferred histone acetyltransferase inhibitor of the present invention is one with the structure 
where the H is [CHR11] is absent if R11 is oxygen
where n is an integer in the range 0 to 2;
where R1, R2, and R10 are independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, pentyl, vinyl, ethinyl, allyl, methyloxy, ethyloxy, propyloxy, butyloxy, and pentyloxy;
where R11 for each R11 is independently selected (e.g., if n is 3, there are three R11 moieties that can be independently selected) from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, pentyl, vinyl, ethinyl, allyl, methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy fluoro, chloro, bromo, iodo, hydroxy, carboxy, and oxygen,
where carboxy is 
wherein R12 is hydrogen, methyl, ethyl, propyl, or isopropyl,
where R8 is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, vinyl, ethinyl, allyl, methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, an amino acid or a polypeptide comprising two amino acids, provided that if R8 is an amino acid or polypeptide, said amino acid or polypeptide may have a protective group (e.g., it is acetylated) at its N terminus. The intent of the protective group is to provide ptrotection during the compound""s synthesis.
Where R9 is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, vinyl, ethinyl, allyl, methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, an amino acid acetylated at its N terminus, or a polypeptide of two or more amino acids; and pharmaceutically acceptable salts thereof.
Inhibitors of interest that are analogs of Lys-CoA are also shown in FIG. 7, (R6 and R7 in FIG. 7 correspond to R8 and R9, respectively.) For H3-20-CoA analogs of interest include those with substitutions in the lysine moiety similar to those shown for Lys-CoA. In addition, truncations and substitutions of the other amino acids in the peptide backbone using combinatorial approaches can be performed to create analogs.
Specific embodiments of interest are:
those wherein R8, in combination with the carbonyl group adjacent to R8, is an amino acid or a polypeptide of two or more amino acids;
those wherein R9 is an amino acid or a polypeptide of two or more amino acids;
those wherein R8, in combination with the carbonyl group adjacent to R8, is an amino acid of a polypeptide of two or more amino acids, and R9 is an amino acid or a polypeptide of two or more amino acids.
Specific inhibitors of p300 acetyltransferase are preferably:
those wherein R8, in combination with the carbonyl group adjacent to R8, is an amino acid, especially where the amino acid is Gly;
those wherein R9 is an amino acid or a polypeptide of 2 or 3 amino acids, especially where R9 is selected from the group consisting of Gly, Gly-Leu, and Gly-Lys-Gly (such that the leftmost amino acid is the one closest to the NH group adjacent to the R9 group); and
those where wherein R8 is methyl and R9 is hydrogen (also referred to as Lys-CoA herein).
Specific inhibitors of PCAF acetyltransferase are preferably:
those wherein R8, in combination with the carbonyl group adjacent to R8, is a polypeptide comprising at least three amino acids, especially where the three amino acids are those of Gly-Gly-Thr and in that sequence;
those wherein R8, in combination with the carbonyl group adjacent to R8, comprises a polypeptide of at least 8 amino acids especially where the eight amino acids are those of Thr-Ala-Arg-Lys-Ser-Thr-Gly-Gly- (SEQ ID NO:1) and in that sequence:
those wherein R9 is a polypeptide of at least 5 amino acids, especially where the 5 amino acids are those of Ala-Pro-Arg-Lys-Gln (SEQ ID NO:2) and in that sequence; and
those where R8 is (N-acetyl)-Ala-Arg-Thr-Thr-Lys-Gln-Thr-Ala-Arg-Lys-Ser-Thr-Gly-NH-CH2- in that sequence, wherein the peptide portion of R8 is SEQ ID NO:3, and R9 is the polypeptide Ala-Pro-Arg-Lys-Gln-Leu (SEQ ID NO:4) in that sequence.